Imaging proteins inside cells with fluorescent tags

Watching biological molecules provides clues to their function and regulation. Some of the most powerful methods of labeling proteins for imaging use genetically encoded fluorescent fusion tags. There are four standard genetic methods of covalently tagging a protein with a fluorescent probe for cellular imaging. These use (i) autofluorescent proteins, (ii) self-labeling enzymes, (iii) enzymes that catalyze the attachment of a probe to a target sequence, and (iv) biarsenical dyes that target tetracysteine motifs. Each of these techniques has advantages and disadvantages. In this review, we cover new developments in these methods and discuss practical considerations for their use in imaging proteins inside living cells.     

Turing patterns inside cells

Concentration gradients inside cells are involved in key processes such as cell division and morphogenesis. Here we show that a model of the enzymatic step catalized by phosphofructokinase (PFK), a step which is responsible for the appearance of homogeneous oscillations in the glycolytic pathway, displays Turing patterns with an intrinsic length-scale that is smaller than a typical cell size. All the parameter values are fully consistent with classic experiments on glycolytic oscillations and equal diffusion coefficients are assumed for ATP and ADP. We identify the enzyme concentration and the glycolytic flux as the possible regulators of the pattern. To the best of our knowledge, this is the first closed example of Turing pattern formation in a model of a vital step of the cell metabolism, with a built-in mechanism for changing the diffusion length of the reactants, and with parameter values that are compatible with experiments. Turing patterns inside cells could provide a check-point that combines mechanical and biochemical information to trigger events during the cell division process.     

Mapping molecular landscapes inside cells

Cryoelectron tomography opens a window into the inner space of cells. It combines the potential of three-dimensional imaging with a close-to-life preservation of biological samples. Tomograms with molecular resolution are essentially images of the cellular proteome and, in conjunction with advanced pattern recognition techniques, they can be used to map the molecular landscape inside organelles and cells.     

An inside look at hippocampal silent cells

In this issue of Neuron, Greenberg and colleagues revise our understanding of how activity-dependent MeCP2 phosphorylation regulates distinct aspects of brain development and circuit function. The study also suggests a prominent role for MeCP2 in the regulation of global chromatin state in?vivo.     

Imaging blood flow inside highly scattering media using ultrasound modulated optical tomography

We report the use of ultrasound modulated optical tomography (UOT) with heterodyne parallel detection to locally sense and image blood flow deep inside a highly scattering medium. We demonstrate that the UOT signal is sensitive to the speed of the blood flow in the ultrasound focus and present an analytical model that relates UOT signals to the optical properties (i. e. scattering coefficient, anisotropy, absorption, and flow speed) of the blood and the background medium. We found an excellent agreement between the experimental data and the analytical model. By varying the integration time of the camera in our setup, we were able to spatially resolve blood flow in a scattering medium with a lateral resolution of 1.5 mm.

     

Development of nitroxides for selective localization inside cells

The use of nitroxides to measure intracellular phenomena, especially oxygen concentrations, is a new and potentially important approach to a number of physiological and pathophysiological studies. This study provides data indicating the feasibility of developing nitroxides that localize selectively in the intracellular compartment; it is based on the use of readily hydrolysed ester linkages, such that the nitroxides become converted intracellularly to ionic derivatives that do not cross cell membranes readily. Up to 120-fold increased concentrations of intracellular nitroxides (and their one electron reduction product, the hydroxylamines) were obtained. The ESR spectra of the intracellular nitroxides were consistent with their conversion to the ionic species. Preliminary studies indicate that these nitroxides have the properties needed for their use as probes of intracellular concentrations of oxygen and that it should be feasible to synthesize nitroxides that will be even more effective for this purpose.     

Micro magnetic tweezers for nanomanipulation inside live cells

This study reports the design, realization, and characterization of a multi-pole magnetic tweezers that enables us to maneuver small magnetic probes inside living cells. So far, magnetic tweezers can be divided into two categories: I), tweezers that allow the exertion of high forces but consist of only one or two poles and therefore are capable of only exerting forces in one direction; and II), tweezers that consist of multiple poles and allow exertion of forces in multiple directions but at very low forces. The magnetic tweezers described here combines both aspects in a single apparatus: high forces in a controllable direction. To this end, micron scale magnetic structures are fabricated using cleanroom technologies. With these tweezers, magnetic flux gradients of nablaB = 8 x 10(3) T m(-1) can be achieved over the dimensions of a single cell. This allows exertion of forces up to 12 pN on paramagnetic probes with a diameter of 350 nm, enabling us to maneuver them through the cytoplasm of a living cell. It is expected that with the current tweezers, picoNewton forces can be exerted on beads as small as 100 nm.     

Photobleaching GFP reveals protein dynamics inside live cells

Cell biologists have used photobleaching to investigate the lateral mobility of fluorophores on the cell surface since the 1970s. Fusions of green fluorescent protein (GFP) to specific proteins extend photobleaching techniques to the investigation of protein dynamics within the cell, leading to renewed interest in photobleaching experiments. This article revisits general photobleaching concepts, reviews what can be learned from them and discusses applications illustrating the potential of photobleaching GFP fusion proteins inside living cells.     

VEGF signaling inside vascular endothelial cells and beyond

Vascular endothelial growth factor-A (VEGF-A) has long been recognized as the key regulator of vascular development and function in health and disease. VEGF is a secreted polypeptide that binds to transmembrane tyrosine kinase VEGF receptors on the plasma membrane, inducing their dimerization, activation and assembly of a membrane-proximal signaling complex. Recent studies have revealed that many key events of VEGFR signaling occur inside the endothelial cell and are regulated by endosomal receptor trafficking. Plasma membrane VEGFR interacting molecules, including vascular guidance receptors Neuropilins and Ephrins also regulate VEGFR endocytosis and trafficking. VEGF signaling is increasingly recognized for its roles outside of the vascular system, notably during neural development, and blood vessels regulate epithelial branching morphogenesis. We review here recent advances in our understanding of VEGF signaling and its biological roles.     

Virus-derived platforms for visualizing protein associations inside cells

Protein-protein associations are vital to cellular functions. Here we describe a helpful new method to demonstrate protein-protein associations inside cells based on the capacity of orthoreovirus protein muNS to form large cytoplasmic inclusions, easily visualized by light microscopy, and to recruit other proteins to these structures in a specific manner. We introduce this technology by the identification of a sixth orthoreovirus protein, RNA-dependent RNA polymerase lambda3, that was recruited to the structures through an association with muNS. We then established the broader utility of this technology by using a truncated, fluorescently tagged form of muNS as a fusion platform to present the mammalian tumor suppressor p53, which strongly recruited its known interactor simian virus 40 large T antigen to the muNS-derived structures. In both examples, we further localized a region of the recruited protein that is key to its recruitment. Using either endogenous p53 or a second fluorescently tagged fusion of p53 with the rotavirus NSP5 protein, we demonstrated p53 oligomerization as well as p53 association with another of its cellular interaction partners, the CREB-binding proteins, within the inclusions. Furthermore using the p53-fused fluorescent muNS platform in conjunction with three-color microscopy, we identified a ternary complex comprising p53, simian virus 40 large T antigen, and retinoblastoma protein. The new method is technically simple, uses commonly available resources, and is adaptable to high throughput formats.     

Autophagy: a regulated bulk degradation process inside cells

Autophagy is a major intracellular degradation/recycling system ubiquitous in eukaryotic cells. It contributes to the turnover of cellular components by delivering portions of the cytoplasm and organelles to lysosomes, where they are digested. Autophagy is mediated by membrane trafficking of unique double-membrane structures, the so-called autophagosomes, which are formed transiently. Moreover, autophagy is dramatically induced under starvation conditions to maintain an amino acid pool so that essential proteins may be synthesized. Recent studies have revealed insights into the molecular basis of membrane dynamics and the regulation of autophagy, which had remained cryptic for a long time.